Introduction

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Nicotine has diverse pharmacological effects on the CNS, many of which are marked by both stimulant and depressant phases of action. These effects include alterations in locomotor activity, hypothermia, convulsions, antinociception and others (for a review, see Martin, 1986). The effects of nicotine presumably occur as a result of its interaction with receptors in the CNS. Recent evidence suggests that these receptors are structurally and functionally diverse (for a review, see Patrick and Luetje, 1993). The electrophysiological and molecular evidence for the existence of multiple functional types of neuronal nicotinic acetylcholine receptors emphasizes the need for reliable probes and ligands with which to study the physiological and functional characteristics of these receptors.

The relatively low enantioselectivity of nicotine has been intriguing for many years (Martin, 1986). On the premise that conformational restraint of nicotine should enhance enantioselectivity, we prepared 1,2,3,5,6,10b-hexahydropyrido[2,3g]indolizine, a rigid nicotine analog that incorporated the N-methyl into a third ring. This analog was chosen because of suggestions that it had some biological activity (Catka and Leete, 1978). Unfortunately, this analog failed to produce nicotine-like effects (Kachur et al., 1986), most likely because the N-methyl was not available for receptor interaction. Therefore, we recently synthesized (±)-cis-2,3,3a,4,5,9b-hexahydro-1-methyl-1H-pyrrolo-[3,2-h]isoquinoline, a BN analog depicted in figure 1, which does not interfere with N-methyl (Glassco et al., 1993). The racemate was resolved into its antipodes for pharmacological evaluation. The racemate and its enantiomers were found to be active in inducing hypomotility and antinociception in mice. The most potent of the three, (+)-BN, has an ED₅₀ of 7.13 µmol/kg for hypomotility and 7.45 µmol/kg for antinociception compared with 4.44 and 4.81 µmol/kg, respectively, for nicotine. However, these compounds and, in particular, the (+)-enantiomer failed to compete for [³H]nicotine binding in rat brain homogenates. In addition, the pharmacological effects of (+)-BN were not blocked by mecamylamine, a noncompetitive nicotinic antagonist. We concluded that either the BN analogs are producing their effects by acting at a nicotinic receptor that is not mecamylamine sensitive or they represent a novel class of nicotinic analgesics. There has been considerable interest in the antinociceptive properties of nicotine for several years as a mean of developing new strategies for pain management (Damaj et al., 1994b; Iwamoto and Marion, 1993; May, 1992; Perkins et al., 1994b). These novel BN analogs may provide new insights into the mechanism of nicotine-induced antinociception, as well as nicotine/receptor interactions, or they may represent a new class of non-nicotinic antinociceptive agents.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structures of nicotine and the BN analog.

The objective of the present study was to establish a more complete pharmacological profile of these analogs to determine whether they share a common mechanism with nicotine. Our aim was to evaluate these analogs in several in vivo and in vitro models that have been used to describe the actions of nicotine. Analogs were evaluated for their ability to produce hypothermia and antinociception in mice through the use of different tests (Aceto et al., 1983; Martin et al., 1990, 1993; Suchocki et al., 1991). The agonist effects of the BN analogs were then tested for their sensitivity to different nicotinic antagonists. Typically, nicotinic antagonists such as mecamylamine and DHE have proven to be critical in identification of nicotinic action. There is evidence that mecamylamine may be acting intracellularly to block the actions of nicotine, whereas antagonists such as DHE may be acting directly at the nicotinic ion channel (Damaj et al., 1993; Martin et al., 1989). In addition, compounds were evaluated in drug discrimination, a model of subjective or stimulus effects that has greater selectivity in identifying nicotine-like activity (Rosecrans, 1989; Stolerman, 1988, 1983).

Acute tolerance to the effects of nicotine develops in animals and humans (Perkins et al., 1994a) and is believed to play an important role in the development and maintenance of dependence to this drug. Tolerance to acute administration of nicotine has been reported for nicotine-induced antinociception (Damaj et al., 1996b), hypomotility (Stolerman et al., 1974) and convulsions (Miner and Collins, 1988). Investigation of the development of acute tolerance to BN and its cross-tolerance with nicotine would help in identifying similar mechanisms of action for these drugs; therefore, we studied the development of acute tolerance to the pharmacological effects of BN after systemic administration in mice, and cross-tolerance experiments with nicotine were performed in the different tests.

A biochemical assay that measures nicotinic agonist-stimulated ⁸⁶Rb⁺ efflux in synaptosomes isolated from mouse brain was also used to evaluate potential nicotinic responses of the bridge analogs. Indeed, several observations suggest that the ⁸⁶Rb⁺ efflux assay measures a response that corresponds to the receptor labeled with [³H]nicotine in binding assays (Marks et al., 1993, 1994). The possibility of the involvement of other nicotinic receptor subtypes was examined by testing the affinity the compounds for neuronal [¹²⁵I]-BGTX binding sites.

Although these behavioral models, coupled with biochemical tests, offer sufficient opportunity for ascertaining nicotinic effects, other neuronal systems and receptors may be involved in the actions of the bridge analogs. For that reason, we examined their affinity for >24 different brain receptors using a NOVASCREEN Drug Discovery and Development Program.

	Methods

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Animals

For the binding and ion flux studies, female C57BL/6J/Ibg mice were bred at the Institute for Behavioral Genetics, University of Colorado. Five mice were housed in each cage and permitted free access to food (Wayne Lab Blox) and water. The vivarium in which the mice were housed was maintained on a 12-hr light/dark cycle (lights on 7:00 A.M.). Animals were 60 to 90 days old when used in the experiments. For the in vivo studies, male ICR mice (20-25 g) and male Sprague-Dawley rats (175-225 g), obtained from Harlan Laboratories (Indianapolis, IN), were used throughout the study. They were housed in groups of six and had free access to food and water.

Drugs

()-Nicotine was obtained from Aldrich Chemical Co. (Milwaukee, WI) and converted to the ditartrate salt as previously described (Aceto et al., 1979). DHE hydrobromide and racemic mecamylamine hydrochloride were gifts from Merck, Sharp and Dohme (West Point, PA). Atropine sulfate was purchased from Research Biochemicals Inc. (Natick, MA). Naloxone was supplied by the National Institute on Drug Abuse (Washington, DC). (±)-BN and its enantiomers were prepared according to the method previously described and characterized as the dihydrobromide salt (Glassco et al., 1993). NaCl, KCl, MgSO₄, CaCl₂, bovine serum albumin (fraction V), CsCl, (+)-nicotine (di-p-toluoyltartrate salt) and tetrodotoxin were purchased from Sigma Chemical Co. (St. Louis, MO). Sucrose and HEPES were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Budget Solve Scintillation Cocktail was purchased from RPI (Mt. Prospect, IL). Carrier-free ⁸⁶RbCl and [³H]L-nicotine (N-methyl-[³H]; specific activity, 78.4 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA) and purified according to the method of (Romm et al., 1990) before use. [¹²⁵I]-BGTX ([¹²⁵I]iodotyrosyl; initial specific activity, 240 Ci/mmol) was purchased from Amersham Life Science (Arlington Heights, IL). Drugs injected to animals were dissolved in physiological saline (0.9% sodium chloride) and given in a total volume of 0.2 ml/100 g b.wt. in rats and 1 ml/100 g b.wt. in mice. All doses were expressed as the free base of the drug.

Intrathecal Injections

Intrathecal injections were performed free-hand between the L5 and L6 lumbar space in unanesthetized male mice (Hylden and Wilcox, 1980). The injection was performed using a 30-gauge needle attached to a glass microsyringe. The injection volume in all cases was 5 µl. The accurate placement of the needle was evidenced by a quick movement of the mouse's tail.

Binding Assays

NOVA screen. BN analog affinity for the receptors and sites adenosine, alpha-1, alpha-2, beta adrenergic, dopamine₁, dopamine₂, -aminobutyric acid_A, -aminobutyric acid_B, histamine₁, histamine₂, serotonin₁, serotonin₂, serotonin₃, cholinergic muscarinic₁, cholinergic muscarinic₂, cholinergic muscarinic₃, phencyclidine (PCP), MK-801, opiates (mu, delta and kappa), cholecystokinin, substance P, substance K and dihydropyridine (L-type calcium channels) was tested through the NIMH/NOVASCREEN Drug Discovery and Development Program (Hanover, MD). Briefly, competitive binding assays were performed in either 250- or 500-µl volumes containing, by volume, 80% receptor preparations, 10% radioligand and 10% test compound/cold ligand (nonspecific binding determinant)/4% dimethylsulfoxide (vehicle). All compounds were solubilized in dimethylsulfoxide, which was diluted to a final concentration of 0.4% in the assay. Assays were terminated by rapid filtration over Whatman glass-fiber filters followed by rapid washing with cold buffer. Radioactivity was determined by liquid scintillation or gamma spectrometry. Data were reduced by a software program proprietary to NOVASCREEN.

[³H]L-Nicotine and [¹²⁵I]-BGTX binding. The inhibition of the binding of [³H]L-nicotine and [¹²⁵I]-BGTX to mouse brain thalamic membranes by ()-nicotine, (+)-nicotine, ()-BN and (+)-BN was determined using modifications of the method of Marks et al. (1986). The binding reactions were conducted at 21°C in 100 µl of buffer (containing 135 mM NaCl, 5 mM CsCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 25 mM HEPES 1/2Na, 20 mM glucose and 0.1%; bovine serum albumin, pH 7.5). In addition to thalamic membranes (100-200 µg), the samples contained either [³H]L-nicotine (11.3 nM) or [¹²⁵I]-BGTX (1.0 nM) and ()-nicotine, (+)-nicotine, ()-BN or (+)-BN at concentrations of 0.3 nM to 3 mM. Total binding was measured in the absence of unlabeled compounds, and nonspecific binding was determined in samples containing 10 µM ()-nicotine (for [³H]L-nicotine) or 1 mM ()-nicotine (for [¹²⁵I]-BGTX). Incubation times were 30 min and 6 hr for [³H]L-nicotine and [¹²⁵I]-BGTX, respectively. The binding reaction was terminated by dilution with ice-cold wash buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄ and 10 mM HEPES 1/2Na, pH 7.5) and filtration onto glass-fiber filters that had been soaked in wash buffer containing 0.5% polyethylenimine (top filter: GB1OOR, Microfiltration Systems, Dublin, CA; bottom filter: Type A/E, Gelman Sciences, Ann Arbor, MI) using a cooled Inotech Cell Harvester (Inotech, Lansing, MI) equipped with a 96-place head. The filtered samples were subsequently washed five times with ice-cold wash buffer. Tritium was measured at 55% efficiency with a Packard 1600 Liquid Scintillation counter using 7-ml vials and 1 ml of Budget Solve Cocktail, and ¹²⁵I was measured at 80% efficiency with a Packard AutoGamma Counter. The K_D and B_max values for both ligands were also determined from saturation curves constructed using eight concentrations of each ligand. Protein was measured according to the method of Lowry et al. (1951) using bovine serum albumin as the standard. The IC₅₀ values for inhibition of [³H]L-nicotine and [¹²⁵I]-BGTX were determined using the equation: Bound_i = bound₀/[l + (I/IC₅₀)]. Inhibition constants were subsequently estimated using the Cheng-Prusoff equation.

⁸⁶Rb⁺ Efflux

The stimulation of ⁸⁶Rb⁺ efflux from crude mouse thalamic synaptosomes by ()-nicotine, (+)-nicotine, ()-BN and (+)-BN and the inhibition of ()-nicotine-stimulated efflux by ()-BN and (+)-BN were measured according to the method of Marks et al. (1993). Crude synaptosomes were prepared by homogenizing the mouse thalamus in 0.32 M sucrose and 5 mM HEPES 1/2Na (pH 7.5), centrifuging the homogenate for 10 min at 1000 × g and centrifuging the resulting supernatant at 10,000 × g for 20 min. The resulting pellet was resuspended in 150 µl incubation buffer per thalamus (incubation buffer contained 140 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM glucose and 25 mM HEPES 1/2Na, pH 7.5). Loading with ⁸⁶Rb⁺ was achieved by incubating a 25-µl aliquot of the synaptosomes with 10 µl of incubation buffer containing 4 µCi of isotope for 30 min. After the incubation with ⁸⁶Rb⁺, the synaptosomes were harvested by filtration onto a 7-mm Gelman A/E glass-fiber filter under gentle vacuum (100 mm Hg). After two washes with 0.5 ml of incubation buffer, the filter containing the synaptosomes was transferred to the apparatus and perfused with 1.5 ml/min experimental buffer (135 mM NaCl, 5 mM CsCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM glucose, 50 nM tetrodotoxin, 0.1% bovine serum albumin and 25 mM HEPES 1/2Na, pH 7.5). After a 6-min wash period, sample collection was begun. Fractions were collected every 30 sec for 6 min. Stimulation of ⁸⁶Rb⁺ efflux by ()-nicotine, (+)-nicotine, ()-BN and (+)-BN was tested by exposing the thalamic synaptosomes to known concentrations of the test compounds for 1 min.

Inhibition of ⁸⁶Rb⁺ efflux was determined by simultaneous exposure of the thalamic synaptosomes to ()-nicotine (0.3, 1.0 or 3.0 µM) and a known concentrations of ()-BN or (+)-BN.

The EC₅₀ values and maximal efflux rates were calculated using the Michaelis-Menten equation: V = V_max * D/(D + K_D). The IC₅₀ values were estimated using the equation: V_i = V_o/[l + (I/IC₅₀)], and apparent K_I values were calculated using the Cheng-Prusoff equation at each ()-nicotine concentration. All curve fits were achieved using the nonlinear least-squares algorithm in Sigma Plot 5.0 (Jandel Scientific, San Rafael, CA).

Behavioral Assays in Mice

Antinociception. Nicotine-induced antinociception in mice was measured by use of the tail-flick method (D'Amour and Smith, 1941; Dewey et al., 1970). Groups of six animals were used for each dose and treatment. A control response (2-4 sec) was determined for each animal before treatment, and test latencies were assessed at various times after drug administration. A maximum latency of 10 sec was imposed if no response occurred within that time. Antinociceptive response was calculated as %MPE), where %MPE = {[(test  control)/(10  control)] × 100}. Mice were pretreated subcutaneously with saline, DHE, mecamylamine, atropine or naloxone 10 min before the BN analogs and tested 5 min later. The doses of nicotinic antagonists used in this protocol are several times higher than the AD₅₀ values determined to block nicotine-induced antinociception in mice (Damaj et al., 1994a).

Body temperature. Rectal temperature was determined with a thermistor probe (inserted 24 mm) and a digital thermometer (Yellow Springs Instrument Co., Yellow Springs, OH). Readings were taken just before and 30 min after the subcutaneous injection of nicotine or the BN analogs. Mice were pretreated with saline, DHE or mecamylamine (subcutaneous) 10 min before nicotine analogs. The difference in rectal temperature before and after treatment was calculated for each mouse. The ambient temperature of the laboratory varied from 21° to 24°C from day to day. Eight to 12 mice were tested in each treatment group, and each animal was tested only once.

Nicotine Drug Discrimination

Subjects. Rats were housed individually in a temperature-controlled environment and provided a diet (Agway Rodent Chow) that maintained their body weight at ~85% of their free feeding weight. Water was available ad libitum in the home cages.

Training procedure. A two-lever, operant drug-discrimination paradigm VI 15 was used for eight operant chambers (four Lafayette model 80001 and four BRS/LVE model sec 002). Reward was a Bioserv 45-mg precision dustless pellet. A microcomputer with Logic 1 interface (MED Associates, Georgia, VT) and MED-PC software (MED Associates) was used to control schedule contingencies and record data. Rats were trained to respond on one lever after a subcutaneous injection of nicotine (0.4 mg/kg) and the other lever after a subcutaneous injection of saline. Rats were placed in the operant chambers 5 min after the injections. The specific procedure for training rats to discriminate nicotine has been previously described (Rosecrans, 1989). Animals were run 5 days/week on a double-alternation schedule (2 days of nicotine and then 2 days of vehicle). Animals learned to discriminate nicotine from vehicle after 50 to 100 training sessions.

Criterion testing. Animals were required to meet a criterion of 3 successive days of 80% correct-lever responding before testing was initiated. The criterion testing sessions lasted 2 minutes and were run under extinction (no reinforcement for correct or incorrect responding). Test sessions were coupled with training sessions of 13 minutes. During the training portion of the session, the animal was reinforced for pressing the appropriate lever for that day's injection. After animals met the criterion, test sessions were conducted on Monday and Thursday. If an animal pressed 80% on the correct lever during a check session, they were tested the next day. This schedule resulted in Wednesday being a training day.

General testing. Test sessions, like check sessions, were 2 minutes long and were run in extinction. However, unlike check sessions, there was no training component.

Dose-response testing. Initial testing was used to assess the dose-responsiveness of nicotine and BN analogs under the VI-15 schedule after subcutaneous injection at different doses. Injections were given 5 min before placement of the animal in the operant chamber. To determine whether BN analogs might alter the discriminative stimulus cue of nicotine, studies were conducted in which the analogs were administered 5 min before nicotine and drug discrimination testing was conducted 5 min after the nicotine injection. The schedule of injections was determined using a Latin-square design.

Statistical Analysis

Data were analyzed statistically by an analysis of variance followed by the Fisher PLSD multiple-comparison test. The null hypothesis was rejected at the .05 level. ED₅₀ values with 95% CLs were calculated by unweighted least-squares linear regression for log-doses probits, as described by Tallarida and Murray (1987). The effects of drugs on rectal temperature were calculated from double-reciprocal analysis (1/effect vs. 1/dose) to yield a theoretical maximum effect, as described by Tallarida and Murray (1987). The ED₅₀ values were determined by calculating the functional response for each drug dose (based on the maximum effect being 1.0), converting the data to probit values and determining the unweighted least-squares linear regression for the log-dose vs. probit as described by Tallarida and Murray (1987). For the PPQ test, ED₅₀ values with 95% CLs were determined according to the method of Litchfield and Wilcoxon (1949).

	Results

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Binding Assays

BN enantiomers at 10 µM had no effect on the binding of radioligands to mu, delta or kappa opioid; muscarinic; serotonin₁, serotonin₂ or serotonin₃; adenosine; adrenergic; dopamine₁ and dopamine₂; -aminobutyric acid; cholecystokinin; substance P; excitatory amino acid; dihydropyridine or PCP receptors (data not shown).

The inhibition of the binding of [³H]L-nicotine by ()-nicotine, (+)-nicotine, ()-BN and (+)-BN is shown in figure 2. Although all four compounds inhibited the binding of [³H]L-nicotine, these compounds differed markedly in potency. ()-Nicotine was the most potent inhibitor, with a K_I value of 3.1 nM, and was ~30-fold more potent than (+)-nicotine (K_I = 99 nM). Both ()-BN, with a K_I value of 23 µM, and (+)-BN, with a K_I value of 39 µM, were much less potent inhibitors of [³H]L-nicotine binding than either nicotine isomer.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]L-nicotine binding. Inhibition of binding to mouse thalamic membranes was measured using 11.3 nM [3H]L-nicotine. Each point represents the mean ± S.E.M. of three different tissue preparations. Average control binding was 63.8 ± 2.6 fmol/mg of protein. The average KD and Bmax values were 3.6 ± 0.6 nM and 82.7 ± 5.2 fmol/mg of protein, respectively.

The inhibition of [¹²⁵I]-BGTX binding by ()-nicotine, (+)-nicotine, ()-BN and (+)-BN is illustrated in figure 3. All four compounds bound in a competitive fashion. ()-Nicotine was the most potent of the four compounds, with a K_I value of 0.21 µM. (+)-Nicotine was approximately one fifth as potent as ()-nicotine (K_I = 1.0 µM). The BN compounds were relatively more potent inhibitors of [¹²⁵I]-BGTX binding than they were of [³H]L-nicotine binding. The inhibitory potency of ()-BN (K_I = 0.98 µM) was nearly identical to that of (+)-nicotine, whereas (+)-BN was approximately one sixth as potent (K_I = 6.2 µM). Both ()-BN and (+)-BN were more potent inhibitors of [¹²⁵I]-BGTX binding than they were of [³H]L-nicotine binding.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Inhibition of [125I]-BGTX binding. Inhibition of binding to mouse thalamic membranes was measured using 1.2 nM [125I]-BGTX. Each point represents the mean ± S.E.M. of six different tissue preparations. Average control binding was 8.2 ± 0.5 fmol/mg of protein. The average KD and Bmax values were 1.1 ± 0.4 nM and 14.8 ± 3.2 fmol/mg of protein, respectively.

⁸⁶Rb⁺ Efflux

Concentration-effect curves for the stimulation of ⁸⁶Rb⁺ efflux from mouse thalamic synaptosomes are shown in figure 4. Both ()-nicotine and (+)-nicotine evoked saturable, concentration-dependent efflux of ⁸⁶Rb⁺. However, ()-nicotine was ~16 times more potent than (+)-nicotine (EC₅₀ = 0.44 ± 0.05 and 7.4 ± 1.4 µM, respectively); ()-nicotine also evoked greater efflux than (+)-nicotine (maximal efflux rates = 2.58 ± 0.07% and 1.96 ± 0.10% tissue content/min, respectively). In contrast, neither () nor (+)-BN stimulated ⁸⁶Rb⁺ efflux at concentrations as high as 100 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Stimulation of 86Rb+ efflux. Concentration-effect curves were measured using mouse thalamic synaptosomes that had been loaded with 86Rb+. Each point represents the mean ± S.E.M. of four samples.

As illustrated in figure 5, both BN analogs were inhibitors of nicotine-stimulated ⁸⁶Rb⁺ efflux. The progressive shift to the right of the inhibition curves as the concentration of ()-nicotine increased indicates that the inhibition is competitive. The IC₅₀ value for ()-BN increased from 3.1 ± 1.4 to 8.1 ± 1.1 to 20.9 ± 3.0 µM as the concentration of ()-nicotine increased from 0.3 to 1.0 to 3.0 µM. Similarly, the IC₅₀ values for (+)-bridged nicotine increased from 22.9 ± 4.8 to 33.0 ± 5.0 to 83.7 ± 18.5 µM. The inhibition constants (K_I values) were subsequently calculated to be 2.4 and 11.5 µM for ()-BN and (+)-BN, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Inhibition of nicotine-stimulated 86Rb+ efflux. Inhibition of 86Rb+ efflux by ()- or (+)-BN was measured at three concentrations of ()-nicotine. Each point represents the mean ± S.E.M. of three samples. Average control efflux was 0.87 ± 0.10, 1.77 ± 0.08 and 2.08 ± 0.18 U at 0.3, 1.0 and 3.0 µM ()-nicotine, respectively.

Discrimination Studies

(±)-BN was administered at doses of 1.2, 2.4 and 4.8 mg/kg and was found to produce severe rate suppression at all three doses. The few responses that were made were predominately on the vehicle lever. Efforts were then made to determine whether the individual enantiomers would produce nicotine-like responding without rate suppression. ()-BN, at doses of 0.4, 0.8, 1.6, 3.2 and 6.4 mg/kg free base, failed to generalize from the nicotine cue but significantly reduced the response rate. However, contrary to (+)-BN, the decrease observed after the ()-isomer was not dose dependent (fig. 6B). (+)-BN also failed to generate nicotine-like responding when tested at doses of 0.1, 0.2, 0.4 and 0.8 mg/kg free base. Severe rate suppression was produced in three of the five rats at 0.4 mg/kg and for all five rats at 0.8 mg/kg (fig. 6B).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Generalization tests with (+)-BN () or ()-BN () in rats trained to discriminate 0.4 mg/kg nicotine from saline. A, Mean ± S.E.M. percent nicotine-correct responding of rats after injection of different doses of BN isomers (in mg/kg subcutaneous) B, Responses per second for dose-response curve for BN isomers in nicotine-trained rats. The effects of saline () and nicotine () (0.4 mg/kg) in trained rats are also represented. Each point represents the mean ± S.E.M. of 5 to 7 rats.

To determine whether these analogs might alter the discriminative stimulus cue of nicotine, studies were conducted in which the analogs were administered 5 min before nicotine and drug discrimination testing was conducted 5 min after the nicotine injection. Because (±)-BN produced severe rate suppression at all three doses, we chose to study the lowest dose of 1.2 mg/kg with combination of nicotine. As can be seen in figure 7A, nicotine produced a dose-related generalization that was the same both before and after testing nicotine in the presence of (±)-BN. As stated above, administration of (±)-BN alone at a dose of 1.2 mg/kg produced severe rate suppression. When this dose of (±)-BN was injected before nicotine, the rate suppressive characteristic of (±)-BN did not occur at the two higher doses of nicotine [response rate of 0.85 ± 0.05 with 0.2 mg/kg nicotine vs. 0.40 ± 0.01 with the combination of 0.2 mg/kg nicotine and 1.2 mg/kg (±)-BN]. Pretreatment with (±)-BN produced a significant potentiation of the effects of nicotine at its lower doses [with 0.2 mg/kg (df = 5, t = 2.943, P < .0321), with 0.1 mg/kg (df = 3, t = 10.521, P < .0018) and with 0.05 mg/kg (df = 4, t = 8.47, P < .0011)], as indicated in figure 7A.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Potentiation of nicotine drug discrimination by (±)-BN and its isomers. A, Percent correct nicotine responding for nicotine alone before (Pre-Dose Response) and after (Post-Dose Response) the combination study in which 1.2 mg/kg (±)-BN was administered 5 min before nicotine. B, Percent correct nicotine responding for nicotine before and after pretreatment with 0.2 mg/kg (+)-BN or 2 mg/kg ()-BN. BN isomers were administered 5 min before nicotine. *, Significantly different from the corresponding dose of nicotine alone (before) at P < .05. The effects of (±)-BN () (1.2 mg/kg) in trained rats are also represented. Each point represents the mean ± S.E.M. of 5 to 7 rats.

To determine which enantiomer was responsible for this augmentation, these interaction studies were replicated with the individual enantiomers. When 2.0 mg/kg ()-BN was given 5 min before administration of nicotine at doses of 0.05 and 0.1 mg/kg, the rats responded as if they had been injected with vehicle. However, administration of the (+)-enantiomer at a low dose (0.2 mg/kg) 5 min before nicotine (0.025 and 0.1 mg/kg) potentiated the discriminative stimulus (fig. 7B). Indeed, responses at the nicotine lever increased from 14 ± 4% to 25 ± 6% in rats pretreated with (+)-BN and challenged with nicotine at 0.025 mg/kg. The response of higher dose of nicotine (0.1 mg/kg) was significantly increased in rats pretreated with (+)-BN (40 ± 10% vs. 60 ± 7% in saline- and (+)-BN-treated rats, respectively). Higher doses of (+)-BN were not tested.

Antinociceptive Studies

Dose-response relationships were established for BN isomers in mice by measuring antinociception at the time of maximal effect (fig. 8) in the hot-plate test. At 5 min after (+)-BN administration (subcutaneous), ED₅₀ values (CLs) were determined to be 3.1 (1.6-5.9) mg/kg or 9 µmol/kg. ()-BN was less active than the (+)-isomer, with 40% analgesia at a dose of 40 mg/kg. When mice were pretreated with (+)-BN, abdominal stretching behaviors were inhibited in a dose-related manner (fig. 8), yielding ED₅₀ values of 1.5 (0.6-4.0) mg/kg. Furthermore, a significant enantioselectivity was found in mice after subcutaneous administration with the ()-isomer in the PPQ test, with a dose of 30 mg/kg yielding a 55% analgesia.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Dose-response relationship of (+)-BN- and ()-BN-induced antinociception in mice after subcutaneous administration in the PPQ and hot-plate (HP) tests. Mice were tested 5 min after the injections. Each point represents the mean ± S.E.M. of 8 to 12 mice.

Antagonism studies. The subcutaneous administration of (+)-BN (6 mg/kg) produced >80% antinociception in the tail-flick test, which was consistent previous reports (Glassco et al., 1993). This effect of (+)-BN was not blocked by pretreatment with several antagonists (fig. 9). Indeed, mecamylamine, a noncompetitive nicotinic antagonist, at doses of 1 and 5 mg/kg did not affect (+)-BN antinociception. A 3 mg/kg dose of DHE, a competitive nicotinic antagonist, also failed to alter the antinociceptive effects of (+)-BN. We previously showed that the AD₅₀ values of these antagonists, when used in this protocol to block nicotine-induced antinociception, were 0.045 and 0.45 mg/kg for mecamylamine and DHE, respectively (Damaj et al., 1994a). In addition, naloxone (1 mg/kg) and atropine (10 mg/kg), opiate and muscarinic receptor antagonists, respectively, failed to block or attenuate (+)-BN-induced antinociception in the tail-flick test. Similarly, mecamylamine (at 1 and 10 mg/kg) and naloxone (at 1 mg/kg) failed to significantly block the effects of (+)-BN in the hot-plate and PPQ tests (data not shown). By themselves, the antagonists did not cause antinociception at the indicated doses and times in all tests.

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Effect of mecamylamine (1 and 5 mg/kg), DHE (3 mg/kg), naloxone (1 mg/kg) and atropine (10 mg/kg) on (+)-BN-induced antinociception in mice. The antagonists were administered subcutaneously 10 min before (+)-BN (6 mg/kg subcutaneous), and the mice were tested 5 min later. Each point represents the mean ± S.E.M. of 8 to 12 mice. DHE (3), 3 mg/kg; MECA (1; mecamylamine), 1 mg/kg; MECA (5), 5 mg/kg; NALX (1; naloxone) (1 mg/kg); ATP) (10; atropine), 10 mg/kg.

Antinociception after intrathecal injection. Intrathecal administration of (+)-BN to mice caused a dose-dependent antinociception with an ED₅₀ value of 102 (46-185) µg/animal or 0.29 µmol/animal (fig. 10). Nicotine was four times more potent than (+)-BN after intrathecal injection [11.8 (7-22) µg/animal or 0.07 µmol/animal[. On the other hand, the effect of an intrathecal dose of 500 µg of ()-BN/animal (12% MPE) was not significantly different from the saline control.

View larger version (11K): [in this window] [in a new window]   Fig. 10.   Dose-response relationship of () nicotine-, () (+)-BN- and () ()-BN-induced antinociception in mice after intrathecal injection in the tail-flick test. The mice were tested 5 min after the intrathecal injections. Each point represents the mean ± S.E.M. of 8 to 12 mice.

Development of tolerance. To determine whether animals could develop acute tolerance to the antinociceptive effects of (+)-BN, mice received a dose of 10 mg/kg (+)-BN and then different groups of animals were challenged at hourly intervals with subsequent doses of 10 mg/kg (+)-BN. Animals were tested only once, and the maximum number of treatments any group received was four subcutaneous injections. Figure 11 shows that tolerance developed to (+)-BN-induced antinociception after the first dose. Indeed, a significant decrease of the %MPE was seen after the second injection of the drug (from 92% to 39%). After the third dose, the effect was no longer significantly different from the saline control. However, no acute cross-tolerance developed to (+)-BN-induced antinociception after subcutaneous administration (table 1) in mice pretreated with nicotine. Indeed, animals pretreated with nicotine developed acute tolerance to a subsequent dose of nicotine but not (+)-BN in the tail-flick test.

View larger version (48K): [in this window] [in a new window]   Fig. 11.   Development of acute tolerance to (+)-BN-induced antinociception in mice. Animals received a dose of 10 mg/kg (+)-BN and then were challenged at hourly intervals with subsequent doses of 10 mg/kg same drug. Animals were tested only once. Each point represents the mean ± S.E.M. of 6 to 8 mice. *, Statistically different from saline (0 dose) at P < .05. §, Statistically different from the single dose of (+)-BN at P < .05.

Body Temperature Studies

Effect on body temperature. The time course of the effect of (+)-BN on body temperature was similar to that of nicotine, with a maximum occurring at 15 to 30 min after injection. At 2 hr after injection, the effect was not significantly different from control values (fig. 12A). (+)-BN after systemic subcutaneous administration caused a dose-dependent hypothermia in mice with an ED₅₀ value of 5.88 (3.2-10.7) mg/kg or 16.3 µmol/kg (fig. 12B). (+)-BN-induced hypothermia was 3 times less potent than the effect of nicotine [ED₅₀ = 1.00 (0.57-1.63) mg/kg or 6 µmol/kg]. ()-BN was less potent in producing hypothermia than the (+)-BN with an ED₅₀ value of 88 (34-228) mg/kg (fig. 12B).

View larger version (32K): [in this window] [in a new window]   Fig. 12.   (+)-BN-induced hypothermia after subcutaneous administration in mice. A, Time course of the effects of () saline (Sal), () nicotine (NIC) and () (+)-BN on body temperature in mice. Nicotine (2 mg/kg) and (+)-BN (5 mg/kg) were given subcutaneously, and the rectal temperature was measured 10, 20, 30, 60 and 120 min after injection. B, Dose-response curves of () nicotine, () (+)-BN and () ()-BN-induced hypothermia in mice after subcutaneous injection. The mice were tested 30 min after injection. Each point represents the mean ± S.E.M. of 8 to 12 mice. C, Effect of mecamylamine (1 mg/kg) and DHE (3 mg/kg) on nicotine- and (+)-BN-induced hypothermia in mice. The antagonists were administered subcutaneously 10 min before nicotine (2 mg/kg subcutaneous) and (+)-BN (6 mg/kg subcutaneous). Mice were tested 30 min later. Each point represents the mean ± S.E.M. of 8 to 12 mice.

Antagonism studies. (+)-BN-induced hypothermia after systemic administration in mice (10 mg/kg) was not blocked by pretreatment with mecamylamine (1 mg/kg subcutaneous) or DHE (3 mg/kg subcutaneous). However, nicotine-induced hypothermia (2 mg/kg subcutaneous) was blocked by both nicotinic antagonists (fig. 12C).

Development of tolerance. Using the same protocol described above for the antinociception studies, tolerance to the hypothermic effect of (+)-BN after systemic administration (10 mg/kg subcutaneous) did not develop after repeated administration of (+)-BN (fig. 13).

View larger version (55K): [in this window] [in a new window]   Fig. 13.   (+)-BN-induced hypothermia after repeated administration in mice. Animals were treated as described in the legend to figure 10. Each point represents the mean ± S.E.M. of 6 to 8 mice. *, Statistically different from saline (0 dose) at P < .05.

	Discussion

Top Abstract Introduction Methods Results Discussion References

It has been known for a long time that nicotine has relatively low enantioselectivity (15-30-fold regarding behavioral effects in animals) (Aceto et al., 1979; Nordberg, 1993). One of the most logical explanations for both (+)- and ()-nicotine exhibiting pharmacological activity is the highly flexible nature of nicotine allows both isomers to assume almost superimposable conformations. Synthesis of rigid analogs, such as BN, should therefore result in a greater degree of enantioselectivity. As we recently described (Glassco et al., 1993), (±)-BN and its enantiomers were found to be effective in inducing hypomotility and antinociception in mice, two effects that are shared by nicotine. Although the (+)-enantiomer of BN was less potent than nicotine with regard to antinociception, it was more potent than its corresponding ()-enantiomer in the different analgesic models used. The latter produced antinociception in the tail flick test that was almost 10-fold less than that produced by the (+)-enantiomer. The two enantiomers also differed in that the ()-enantiomer failed to produce motor impairment at doses that were 20 times greater than that of the (+)-enantiomer. The fact that one of the isomers produced pharmacological effects similar to those of nicotine was not unexpected in that these derivatives are structurally related to nicotine. However, the observation that these analogs competed for [³H]nicotine receptor binding only at very high concentrations was surprising. We had also expected the enantioselectivity to be considerably higher. Therefore, it became important to establish the complete pharmacological profile of BN and establish whether it is producing its effects through either nicotinic or non-nicotinic mechanisms. Such an investigation is complicated by molecular and biochemical data that suggest the existence of multiple nicotinic receptors in the CNS to which specific ligands and probes are of limited availability. The present work, combining behavioral and biochemical observations, indicates that nicotinic analogs with intriguing profile are emerging.

It is well established that nicotine drug discrimination is an exceptionally reliable method for assessing nicotine behavioral properties (Stolerman, 1990). The nicotine discrimination has been shown to be highly specific and mediated by central nicotinic receptors. However, the specific receptor subtype mediating this effects is still unknown. It has been suggested that the alpha-4, beta-2 receptor may be involved in the discriminative stimulus of nicotine because an excellent correlation has been found between potency in nicotine drug discrimination and [³H]nicotine binding (Damaj et al., 1996a; Stolerman et al., 1995). Only those compounds that inhibit [³H]nicotine binding sites with high affinity produce nicotine-like discriminative effects. Furthermore, MLA, an alpha-7 antagonist, failed to antagonize the discriminative stimulus of nicotine after systemic and intracerebroventricular administration in rats (Brioni et al., 1996). BN analogs failed to generalize to nicotine at the doses tested. The (+)-enantiomer appears to be slightly more potent than nicotine in producing rate suppression and >10 times more potent than the ()-enantiomer. Clearly, these compounds are entering the brain and producing pharmacological effects. However, the failure of both enantiomers of BN to generalize from the nicotine cue demonstrated that these agents do not share all of the pharmacological properties of nicotine. On the other hand, BN analogs produced hypothermia and antinociception after administration in mice. One logical conclusion is that the analogs are producing these nicotine-like effects by acting through mechanisms distinct from those of nicotine. In addition, the failure of mecamylamine and DHE to antagonize the antinociceptive and hypothermic effects of (+)-BN are consistent with this notion. It is quite evident that constructing this rigid analog resulted in a unique derivative in that it is capable of producing some nicotine-like properties that are believed to be mediated through the alpha-4, beta-2 receptor, yet it does not interact with this receptor in a fashion comparable to that of nicotine.

Although one reasonable conclusion could be that the enantiomers of BN are incapable of interacting with the alpha-4, beta-2 receptor, the potentiation of the nicotine cue in the drug discrimination paradigm suggests otherwise. One possible explanation for the enhancement by BN of the nicotine cue is that it acts at the alpha-4, beta-2 receptor but in such a manner that it fails to directly compete for nicotine. However, the inability of the BN analogs to stimulate ⁸⁶Rb⁺ efflux in brain synaptosomes provide further evidence that the effects produced by the BN analogs are not mediated by the nicotine receptors that are labeled by [³H]nicotine. Actually, the BN analogs were weak inhibitors of nicotine-stimulated ⁸⁶Rb⁺ efflux, an action that is opposite that found in nicotine drug discrimination. It is easier to envision a binding site different from the agonist site to explain the antagonistic activity of BN analogs that do not bind directly to [³H]nicotine sites; however, it is difficult to reconcile potentiating effects in drug discrimination and antagonistic properties in nicotine-stimulated ⁸⁶Rb⁺ efflux.

The lack of agonist effects on the alpha-4, beta-2 receptor subtype, raises the possibility of the involvement of other nicotinic subunits in the actions of BN. It is unlikely that alpha-7 subunits mediate the effects of BN analogs because their affinity to neuronal [¹²⁵I]-BGTX binding sites is in the higher micromolar range. Other nicotinic receptor subtypes remain as possible candidates. Indeed, BN is may be producing its effects by acting on nicotinic receptor subtypes that are not mecamylamine sensitive.

These BN analogs may represent a novel class of analgesics. (+)-BN elicited antinociception in the tail-flick test with an ED₅₀ value of 7.45 and 0.29 µmol/kg after systemic and intrathecal injections, respectively. Its effect is enantioselective because ()-BN produced little analgesia at a dose 5 times higher than the ED₅₀ value for (+)-BN. Similar results were observed with the hot-plate test and a chemically induced pain model or PPQ test, with (+)-BN more active in the PPQ test than in the thermal stimulus tests. The failure of naloxone and atropine to antagonize the antinociceptive effects of (+)-BN suggests that the opiate and muscarinic mechanisms are not involved, which correlates with a lack of affinity of BN analogs to opioid and muscarinic receptors. Finally, a significant degree of tolerance was observed to the antinociceptive effects after repeated administration of (+)-BN, a well known and described phenomenon for nicotine (Ochoa et al., 1990; Tripathi et al., 1982). On the other hand, failure of (+)-BN and nicotine to develop cross-tolerance again suggests separate mechanisms. The fact that (+)-BN was incapable of producing tolerance to its hypothermic effects provides an additional distinction from nicotine.

The overall results of this study demonstrate the complexity of the interaction between nicotinic ligands with their receptors. In this context, BN may represents a family of new nicotinic ligands interacting in a selective manner with nicotinic receptors and may serve as a unique tool for unraveling the complexities of neuronal nicotinic receptors. On the other hand, these novel analogs could be producing their effects via mechanisms not shared directly by nicotine. These results suggest that the acute effects of BN analogs are not mediated by the alpha-4, beta-2 receptor subunit combination. Although the alpha-4, beta-2 receptor is unlikely to mediate the agonist effects of BN, our findings clearly show that BN is acting through a unique mechanism to modulate some of the effects of nicotine.